Aurora A and Aurora B activities do not influence the timing of CENP-A assembly. (A) A nascent pool of CENP-A-SNAP was pulse labeled in randomly cycling HeLa cells during which either Aurora A or Aurora B activity was inhibited by treatment with 1µM of MLN8054 or 2µM of ZM447439, respectively. Cells were fixed and counterstained for cyclin B1, CENP-T and with DAPI to indicate G2 status, centromeres and DNA, respectively. Effective kinase inhibition was evident from chromosome segregation defects in mitosis after Aurora A inhibition [indicated by an asterisk and (Hoar et al., 2007)] prior to subsequent CENP-A assembly in G1 or after Aurora B inhibition resulting in cytokinesis failure and multinucleated cells [asterisk and (Ditchfield et al., 2003)]. Representative images of cells in G1 (low cyclin B) and G2 phase (high cyclin B) are shown. (B) Experiment as in A except that cells were treated for one hour with either Roscovitine alone or in combination with MLN8054 or ZM447439 prior to fixation. Percentage of cells assembling CENP-A-SNAP is indicated [either percent of total cells (in top panel, G1 phase) or percent of cyclin B1 positive cells (bottom panel, G2 phase)].

Dev Cell 2012 22, 52-63. MLN8054 purchased from Selleck

Stacked column graphic representing the effect of the small molecule MLN8054 throughout oocyte developmental progress, namely during metaphase I, telophase I and metaphase II oocytes in different conditions.

MLN8054 Mechanism

Aurora kinase A, also known as Aurora-2, is associated with centrosome maturation and separation and thereby regulates both spindle assembly and stability. Aurora kinase A contains a N-terminal domain, a protein kinase domain and a short C-terminal domain. Like other protein kinases, the catalytic domain of Aurora kinases A is conserved, while the N-terminal domain of Aurora A is less conserved, which determines selectivity during protein-protein interactions. [1] Aurora kinase A catalytic domain has the typical bi-lobal kinase fold, which comprises of an N-terminal lobe and a large C-terminal lobe. The Active site is located at the interface between the lobes and includes the ATP binding site, the catalytic base (Asp256), and the kinase activation loop. [2] Aurora A kinase activity depends on autophosphorylation of Thr288 in the activation loop.
MLN8054 has a benzazepine core scaffold with a fused amino pyrimidine ring and an aryl carboxylic acid. [3] The crystal structure indicates that MLN8054 binds in the ATP binding pocket. Specifically, the amino pyrimidine ring of MLN8054 contacts the hinge region by forming two H-bonds to the backbone of Ala213. Besides, the N-Hs of Arg137 side chain also form two H-bonds to the carbonyl of aryl carboxylic acid. In addition, there exists van de Waals and hydrophobic interactions between MLN8054 and side chains that are in proximity to MLN8054, including Arg137, Lys162, Leu210 (the gatekeeper residue), Tyr212, Ala 213, Gly216, and Thr217.[4]
References
[1] Bolanos-Garcia VM. Int J Biochem Cell Biol. 2005, 37(8), 1572-1577.
[2] Zhao B, et al. Protein Sci. 2008, 17(10), 1791-1797.
[3] Manfredi MG, et al. Proc Natl Acad Sci U S A. 2007, 104(10), 4106-4111.
[4] Sloane DA, et al. ACS Chem Biol. 2010, 5(6), 563-576.

Human tumor cell lines are grown in 96-well cell culture dishes according to the distributor's recommendations. MLN8054, diluted in DMSO, is added to the cells in 2-fold serial dilutions to achieve final concentrations ranging from 10 mM to 0.04 mM. MLN8054 at each dilution is added in triplicate with each replicate on a separate plate. Cells treated with DMSO (n = 6 wells per plate; 0.2% final concentration) serves as the untreated control. The cells are treated with MLN8054 for 96 hours at 37 °C in a humidified cell culture chamber. Cell viability in each cell line is measured by using the Cell Proliferation ELISA, BrdU colorimetric kit according to the manufacturer's recommendation

HCT-116 and PC-3 cells are injected s.c. into the right flank of nude mice.

Formulation

MLN8054 is dissolved in 10% hydroxypropyl-β-cyclodextrin with 5% sodium bicarbonate.

Dosages

≤30 mg/kg

Administration

Administered via p.o.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species

Mouse

Rat

Rabbit

Guinea pig

Hamster

Dog

Weight (kg)

0.02

0.15

1.8

0.4

0.08

10

Body Surface Area (m2)

0.007

0.025

0.15

0.05

0.02

0.5

Km factor

3

6

12

8

5

20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by

Animal B Km

Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

* <1 mg/ml means slightly soluble or insoluble.* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Y-27632 2HCl is a selective ROCK1 (p160ROCK) inhibitor with Ki of 140 nM in a cell-free assay, exhibits >200-fold selectivity over other kinases, including PKC, cAMP-dependent protein kinase, MLCK and PAK.

Crizotinib (PF-02341066) is a potent inhibitor of c-Met and ALK with IC50 of 11 nM and 24 nM in cell-based assays, respectively.

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

VX-680 (Tozasertib, MK-0457) is a pan-Aurora inhibitor, mostly against Aurora A with Kiapp of 0.6 nM in a cell-free assay, less potent towards Aurora B/Aurora C and 100-fold more selective for Aurora A than 55 other kinases. Phase 2.

ZM 447439 is a selective and ATP-competitive inhibitor for Aurora A and Aurora B with IC50 of 110 nM and 130 nM, respectively. It is more than 8-fold selective for Aurora A/B than MEK1, Src, Lck and has little effect against CDK1/2/4, Plk1, Chk1, etc.

MK-5108 (VX-689) is a highly selective Aurora A inhibitor with IC50 of 0.064 nM in a cell-free assay and is 220- and 190-fold more selective for Aurora A than Aurora B/C, while it inhibits TrkA with less than 100-fold selectivity. Phase 1.